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Directed evolution of Clec12a-binding peptides using mammalian-expressed recombinant protein
Yoshirou Kawaguchi1, Md Samiul Islam1,2, Mina Yokoyama1
1Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka, 819-0395, Japan.
Biochemistry and Biophysics Reports
|March 9, 2026
Summary
Researchers developed a scalable mammalian expression system to produce the Clec12a extracellular domain for immune research. This system enabled the discovery of novel peptide binders targeting Clec12a-expressing dendritic cells for immunomodulatory applications.
Area of Science:
- Immunology
- Molecular Biology
- Biochemistry
Background:
- C-type lectin receptors (CLRs) like Clec12a play crucial roles in immune homeostasis.
- Clec12a, expressed on myeloid cells, transmits inhibitory signals, contributing to immune regulation.
- Previous production of murine Clec12a extracellular domain lacked established suitability for ligand discovery and scalability data.
Purpose of the Study:
- To establish a scalable mammalian expression system for the murine Clec12a extracellular domain (mClec12a ECD).
- To utilize the produced mClec12a ECD as bait for directed evolution to discover novel peptide binders.
- To validate the binding and potential application of discovered peptides for targeting Clec12a-positive cells.
Main Methods:
- Established a mammalian expression system yielding approximately 80 mg/L of mClec12a ECD.
- Incorporated an interleukin-2 signal peptide for efficient secretion from Expi293 cells.
- Employed ribosome display technology with mClec12a ECD bait to isolate binding peptides, followed by affinity determination and primary cell binding assays.
Main Results:
- Reproducibly obtained high yields of purified mClec12a ECD with proper N-glycosylation, indicating correct protein maturation.
- Successfully isolated three mClec12a binding peptides (CBPs), with CBP2 exhibiting the highest affinity (KD ≈ 6.29 μM).
- Demonstrated that CBP2 binds to primary dendritic cells, confirming the functional integrity of the recombinant mClec12a used in selection.
Conclusions:
- The developed mammalian expression system provides a scalable and reliable source of functional mClec12a ECD for ligand discovery.
- The identified peptides, particularly CBP2, represent promising first-generation ligands for targeting Clec12a-positive cells.
- These findings lay the groundwork for developing targeted immunomodulatory therapies utilizing Clec12a-specific ligands.
